This application is based upon and claims the benefit of priority from the prior Japanese Patent Applications No. 11-078281, filed Mar. 23, 1999; No. 11-080026, filed Mar. 24, 1999; No. 11-080028, filed Mar. 24, 1999; and No. 11-080202, filed Mar. 24, 1999, the entire contents of which are incorporated herein by reference.
This invention relates to a confocal microscope adapted to observe and measure the micro-structure and the three-dimensional profile of a specimen by utilizing light.
Known typical confocal microscopes adapted to operate at high speed include those comprising a Nipkow""s disk having a large number of pin holes arranged helically at intervals about ten times as large as their diameter. A confocal microscope comprising a Nipkow""s disk is required to eliminate cross talk arising from adjacently located pin holes, and hence relatively large intervals have to be used in order to separate the pin holes from each other. The large intervals reduce the efficiency of utilizing the beam of light from the light source and, as a matter of fact, only 1% of the beam coming from the light source is utilized for the operation of the microscope. This means that the obtained image of the specimen is very dark.
R. Juskaitis, T. Wilson et al. proposed an improvement to confocal microscopes comprising a disk in xe2x80x9cEfficient real-time confocal microscopy with white light sourcesxe2x80x9d, Nature, Vol. 1, 383, October, 1996, pp. 804-806 and International Disclosure No. WO97/31282. FIG. 1 of the accompanying drawings schematically illustrates a confocal microscope as proposed by T. Wilson et al.
Referring to FIG. 1, an optical lens 4 and a half mirror 6 are arranged on the optical path of the beam of light emitted from light source 2, which may be a halogen light source or a mercury light source. A rotary disk 8, an objective lens 10 and a specimen 12 are arranged on the optical path of the light beam reflected by the half mirror 6.
Now, referring to FIG. 2, the rotary disk 8 has a random pin hole pattern section 8a where pin holes are randomly arranged and an aperture section 8b where light can pass freely. The random pin hole pattern section Ba and aperture section 8b are separated from each other by a pair of light blocking sections 8c, 8d that block any light trying to pass therethrough. The rotary disk 8 is linked to the shaft of a motor (not shown) by way of rotary shaft 14 so that it can be driven to rotate at a predetermined constant rate.
The beam of light reflected by the specimen 12 is made to enter CCD camera 18 by way of the objective lens 10, the rotary disk 8, the half mirror 6 and condenser lens 16. The CCD camera 18 is controlled for the timing of its image pickup operation in synchronism with the rotary motion of the rotary disk 8 in such a way that it picks up two images of the specimen getting to it by way of the random pin hole pattern section 8a and the aperture section 8b respectively.
The images output from the CCD camera 18 are stored in computer 20. Of these, the image caught by the camera by way of the random pin hole pattern section 8a is a confocal image to which a non-confocal image (hereinafter referred to as composite image) is overlaid due to the fact that the density of pin holes is about ten times as high as that of pin holes of an ordinary Nipkow""s disk.
Only a confocal image is obtained from subtraction of a composite image containing a confocal component and a conventional image obtained through the aperture section 8b. The calculated confocal image is displayed on the monitor 22.
While only 0.5 to 1% of the beam coming from the light source is utilized in a Nipkow""s disk type confocal microscope, 25 to 50% of the beam coming from the light source is utilized in a confocal microscope proposed by T. Wilson et al. so that they report that an image much clearer and brighter than an image obtained by a conventional Nipkow""s disk type confocal microscope can be obtained by their camera.
Meanwhile, N. A. A. Neil, T. Wilson and R. Juskaitis, xe2x80x9cA Light Efficient Optically Sectioning Microscopexe2x80x9d, Journal of Microscopy, Vol. 189, pt. 2, (1998), pp. 114-117, describes an arrangement as shown in FIG. 3 that is obtained by replacing the disk having randomly arranged pin holes of a known confocal microscope with a disk 24 having a linear pattern section 24a where a large number of light blocking areas and light transmitting areas (slits) are arranged linearly and alternately and an aperture section 24b where light can pass freely, the linear pattern section 24a and the aperture section 24b being separated by a pair of light blocking sections 24c, 24d adapted to block any light trying to pass therethrough. The authors claim that the proposed arrangement using such a disk can also provide a confocal image.
However, the above listed known disk scanning type confocal microscopes are accompanied by the following drawbacks.
While the disk scanning type confocal microscope proposed by T. Wilson et al. (International Disclosure No. WO 97/31282) provides an image tens of several times clearer and brighter than an image that can be obtained by a known Nipkow""s disk type confocal microscope, the former is required to subtract the conventional image obtained by way of the light transmitting areas from the image (composite image) obtained by overlaying a non-confocal image to a confocal image getting to it by way of the pin hole section hole section.
However, the ratio of the brightness of the confocal image component to that of the non-confocal image component of a composite image varies as a function of the density of pin holes and the numerical aperture (NA) of the objective lens. On the other hand, the relationship between the brightness of the non-confocal image component of a composite image obtained by the random pin hole pattern section and that of the conventional image obtained at the aperture is not known. Therefore, it is difficult to obtain an optimal confocal image.
Additionally, a disk having linear slits as light transmitting areas does not provide any confocal image components in the direction parallel to the slits of the pattern but it does in the direction perpendicular to the slits of the pattern. In other words, the confocal effect of such a confocal microscope can vary depending on the direction of the image relative to the linear slits.
Still additionally, there may be cases where it is desirable to allow the non-confocal image to remain to a slight extent in addition to the obtained confocal image in order to vertically observe the specimen. However, with any known confocal microscopes adapted to obtain the confocal image by way of a subtracting process, the effect of the latter is automatically defined by the ratio of the area of the pin hole section and that of the light transmitting section (aperture section) so that the desk has to be replaced in order to change the effect of subtracting the conventional image from the composite image.
In view of the above described problems of known confocal microscopes, it is therefore the first object of the present invention to provide a confocal microscope that can make the brightness of the non-confocal image component of the composite image and that of the conventional image substantially equal to each other in order to obtain an optimal confocal image.
The second object of the present invention is to provide a confocal microscope comprising a rotary disk having a light transmitting section (aperture section) formed by alternately arranging linear light blocking areas and light transmitting areas (slits), the rotary disk being adapted to obtain a relatively uniform confocal image.
The third object of the present invention is to provide a confocal microscope comprising a rotary disk and adapted to vary the ratio of the confocal image component to the non-confocal image component.
According to the invention, the above first object is achieved by providing a confocal microscope comprising:
a lighting means for illuminating a specimen with a beam of light;
an extraction means having sites for transmitting the beam of light emitted from the lighting means and sites for blocking light and adapted to extract a composite image obtained by overlaying a non-confocal image on a confocal image and a conventional image from the beam of light coming from the specimen;
an image pickup means for selectively picking up the composite image and the conventional image extracted by the extraction means; and
a control means for obtaining a confocal image of the specimen from the composite image and the conventional image picked up by the image pickup means, wherein
the extraction means has semi-transmissive regions showing a light transmissivty of k and an aperture region freely transmitting light irradiated from the lighting means, the semi-transmissive regions and the aperture region being adapted to selective use, and the area of the aperture region is equal to that of any of the semi-transmissive regions multiplied by k2.
According to the invention, the above second object is achieved by providing a confocal microscope comprising:
a lighting means for illuminating a specimen with a beam of light;
an extraction means having sites for transmitting the beam of light emitted from the lighting means and sites for blocking light and adapted to extract a composite image obtained by overlaying a non-confocal image on a confocal image and a conventional image from the beam of light coming from the specimen;
an image pickup means for selectively picking up the composite image and the conventional image extracted by the extraction means; and
a control means for obtaining a confocal image of the specimen from the composite image and the conventional image picked up by the image pickup means, wherein
the extraction means is formed by a disk rotatable around a rotary shaft located at the center thereof and the semi-transmissive regions contain a plurality of linear slits allowing light to pass therethrough, the semi-transmissive regions of the disk having a contour of a sector with a central angle not smaller than 90xc2x0, the top of the sector being located at the rotary shaft.
According to the invention, the above third object is achieved by providing a confocal microscope comprising:
a lighting means for illuminating a specimen with a beam of light;
an extraction means having sites for transmitting the beam of light emitted from the lighting means and sites for blocking light and adapted to extract a composite image obtained by overlaying a non-confocal image on a confocal image and a conventional image from the beam of light coming from the specimen;
an image pickup means for selectively picking up the composite image and the conventional image extracted by the extraction means; and
a control means for obtaining a confocal image of the specimen from the composite image and the conventional image picked up by the image pickup means, wherein
the arithmetic operation means carries out a subtraction on the composite image data and the conventional image data obtained by the image pickup means by using a coefficient for realizing a desired ratio.
According to the invention, the above fourth object is achieved by providing a confocal microscope comprising adapted to focus a beam of light by way of a mask pattern member variably operating with a predetermined pattern and an objective lens and cause the beam of light reflected by the specimen to enter an image pickup means by way of the objective lens and the mask pattern member to produce an image of the specimen for observation, the microscope comprising:
a drive means for driving the image pickup means for an image pickup operation in synchronism with the variable operation of the mask pattern member and modifying the relative distance between the objective lens and the specimen along the optical axis of the objective lens.
According to the invention, the above fifth object is achieved by providing a confocal microscope comprising:
a lighting means for illuminating a specimen with a beam of light;
a plurality of objective lenses with different respective magnifications for focusing the beam of light coming from the lighting means and the specimen;
a rotary member having a plurality of pattern sections arranged respectively corresponding to the plurality of objective lenses for obtaining confocal image data of an image including those of the non-confocal component thereof and an aperture section for obtaining non-confocal image data containing only those of the non-confocal component;
a rotary drive means for driving the rotary member to rotate in a predetermined sense;
an image pickup means for picking up an image by means of the beam of light passing through each of the pattern sections and the aperture section of the rotary member driven to rotate by the rotary drive means;
an image processing means for storing the data of each image obtained by the image pickup means and obtaining a confocal image;
a synchronizing signal generating means for generating a synchronizing signal in synchronism with the operation the image pickup means;
a detection means for detecting the state of rotation of the rotary member;
a control means for synchronizing the phase of the detection signal from the detection means and the signal from the synchronizing signal generating means; and
a trigger signal generating means for generating a signal to be used for controlling the image pickup means on the basis of the timing of the signal from the synchronizing signal generating means and the detection signal.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.